MITOCHONDRIA!. OUTER MEMBRANE PERMEABILIZATION IN APOPTOSIS For ten years we have known that mitochondria are central to those forms of apoptosis most relevant to oncogenesis and cancer therapy, and for most of that time we have understood that the Bcl-2 family proteins are integral to this mitochondrial pathway. An emerging view, central to this project is that the pro-apoptotic proteins Bax and Bak, upon activation, induce mitochondrial outer membrane permeabilization (MOMP) leading to the release of proteins of the intermembrane space, such as cytochrome c, that orchestrate caspase activation. This is inhibited by the anti-apoptotic Bcl-2 family members, thereby blocking cell death. Despite this understanding, the precise mechanisms whereby Bax permeabilizes the outer mitochondrial membrane remain obscure. In this proposal, we seek to determine how Bax-mediated membrane events set the process in motion, and explore the nature of this permeabilization and how it is controlled. The questions that follow from these considerations are our specific aims, as follows: 1. Is the activation of Bax or Bak necessary and sufficient for MOMP? Here, we will explore the MOMP event in vivo, in single cells over time, and determine the nature of the signals that are necessary and sufficient for this event. Using a combination of in vitro and in vivo approaches, we will watch this process during apoptosis and determine the relationship of MOMP to other measurable events in the cell, in particular, Bax activation. 2. What is the nature of the Bax-mediated MOMP? We will apply the technique of cryo-electron microscopy to probe the nature of Bax-mediated permeabilization in membranes composed of mitochondrial lipids, using recombinant monomeric Bax activated with specific BH3 peptides. This will mimic the process of MOMP in vivo and provide fundamental insights into how Bax permeabilizes membranes. 3. Why does Bax induce MOMP and apoptosis while BcI-xL is protective? Despite similarities in sequence and structure, Bax and Bcl-xL have opposite functions. Using an unbiased approach, we will determine those regions of each that confer these distinct activities, and compare effects of randomly generated chimeras of the two on yeast and mammalian cell survival, Bax oligomerization, MOMP, and membrane permeabilization. Our project is intimal to the goals of the overall program, which seek to identify key signaling events in apoptosis, in our case focusing on a fundamental step in the process and investigating it in the context of the apoptotic pathway. Our aims are mutually supporting but not inter-dependent, that is, successful completion of one is not necessary for progress in the others. Nevertheless, by approaching this problem from complementary directions we will obtain a deep understanding of this key step and thereby of the mitochondrial pathway in general. Our efforts are ambitious, but are based on sound and consistent progress. Further, our proposed research involves extensive collaboration made possible by the ongoing Program Project.